Today, many pharmacologically active drugs can be effective in vivo only if they are able to achieve and maintain therapeutic concentrations at the site of action. Pharmaceutical properties such as solubility, partition coefficient, permeability, and protein binding contribute to in vivo disposition and, frequently, these properties are important determinants of clinical outcome. The recent successes of combinatorial chemistry in accelerating drug discovery have also increased the interest in rapid, resource-sparing approaches to determining pharmaceutical properties.
The binding of drugs to serum proteins is particularly important, because it affects both the activity of drugs and their disposition. According to the “free drug” hypothesis, only unbound drug exerts pharmacological activity and disposition is often altered by drug binding. Consequently, it is important to know the affinity of a drug for serum proteins.
An example of such a drug is warfarin which is a vitamin K antagonizing anticoagulant derived from coumarin. Warfarin is a clinically important drug widely used in the treatment of thrombolic disorders such as heart attacks and stroke. The mechanism of action of this drug is based on an inhibition of the enzyme vitamin-K dependent reductase (VKOR) which is important for the coagulation of blood. When introduced into blood plasma, 99% of warfarin is reported to be bound to the blood plasma transport protein, human serum albumin (HSA) (Yacobi et al., Clin. Pharmcol. Ther. 1976, 19, 552-558). On account of the fact that HSA demonstrates polymorphism, and that the therapeutic window of the drug is very narrow, careful monitoring of the effect of drug dosage must be performed.
Moreover, other factors have been shown to impact upon the anticoagulant effect of warfarin, e.g. food intake and metabolic rates. Currently, the inhibition of VKOR by warfarin is measured by an indirect method in which the clotting time (prothrombin time) is measured. As self-monitoring with this method is problematic, the development of alternative methods, ideally both more robust and more sensitive, for determination of an amount or concentration of e.g. warfarin present in a patients blood is desirable.
It should be noted that other techniques have been proposed for protein binding measurements including dialysis, ultrafiltration, circular dichroism, and extrinsic fluorescence.